Biogenesis of selected abiotic-stress responsive plant microRNAs

197 Pages·2016·19.92 MB·English

by  Katarzyna Knop

Adam Mickiewicz University in Poznan
Faculty of Biology
Department of Gene Expression
Uniwersytet im. Adama Mickiewicza w Poznaniu
Wydział Biologii
Zakład Ekspresji Genów

Katarzyna Agata Knop

Biogenesis of selected abiotic-stress responsive plant microRNAs
Biogeneza wybranych mikroRNA związanych z odpowiedzią rośliny na stresy abiotyczne

PhD thesis
Rozprawa doktorska

Supervisor: prof. dr hab. Zofia Szweykowska-Kulinska
Promotor: prof. dr hab. Zofia Szweykowska-Kulińska

Poznan, Poland 2017
Poznań, Polska 2017

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FUNDING
This work was supported by the Polish National Science Center NCN
(PRELUDIUM: 2012/05/N/NZ2/00955 and ETIUDA: 2015/16/T/NZ1/00022)
and by the Polish Ministry of Science and Higher Education (for the KNOW RNA
Research Center in Poznan (01/KNOW2/2014)).

PUBLICATIONS
Results presented within this PhD thesis have been published in the following paper:
1. Knop K*, Stepien A*, Barciszewska-Pacak M, Taube M, Bielewicz D, Michalak
M, Borst J W, Jarmolowski A, Szweykowska-Kulinska Z. (2016). Active 5' splice
sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from
intron-containing genes. Nucleic Acids Res doi: 10.1093/nar/gkw895.
In addition, the following papers concerning the biogenesis and function of miRNAs have
been published:
1. Stepien A*, Knop K*, Dolata J*, Taube M, Bajczyk M, Barciszewska-Pacak M,
Pacak A, Jarmolowski A, Szweykowska-Kulinska Z. (2016). Post-transcriptional
coordination of splicing and miRNA biogenesis in plants. Wiley Interdiscip Rev
RNA doi: doi: 10.1002/wrna.1403.
2. Barciszewska-Pacak M, Knop K, Jarmolowski A, Szweykowska-Kulinska Z.
(2016). Arabidopsis thaliana microRNA162 level is posttranscriptionally
regulated via splicing and polyadenylation site selection. Acta Biochim Pol epub,
No. 2016_1349, doi: 10.18388/abp.2016_1349.
3. Barciszewska-Pacak M, Milanowska K, Knop K, Bielewicz D, Nuc P, Plewka P,
Pacak AM, Vazquez F, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z.
(2015). Arabidopsis microRNA expression regulation in a wide range of abiotic
stress responses. Front Plant Sci 6:410.
4. Zielezinski A*, Dolata J*, Alaba S*, Kruszka K*, Pacak A*, Swida-Barteczka A,
Knop K, Stepien A, Bielewicz D, Pietrykowska H, Sierocka I, Sobkowiak L,
Lakomiak A, Jarmolowski A, Szweykowska-Kulinska Z, Karlowski WM. (2015).
mirEX 2.0 - an integrated environment for expression profiling of plant
microRNAs. BMC Plant Biol 15:144.

*equal contribution

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ŹRÓDŁA FINANSOWANIA
Niniejsza praca doktorska powstała dzięki dofinansowaniu w ramach grantów
uzyskanych z Narodowego Centrum Nauki NCN (PRELUDIUM: 2012/05/N/NZ2/00955
i ETIUDA: 2015/16/T/NZ1/00022) oraz ze środków Ministerstwa Nauki i Szkolnictwa
Wyższego (dotacja KNOW dla Poznańskiego Konsorcjum RNA (decyzja numer
01/KNOW2/2014)).

PUBLIKACJE
Wyniki badań objętych niniejszą pracą doktorską zostały przedstawione w artykule:
1. Knop K*, Stępień A*, Barciszewska-Pacak M, Taube M, Bielewicz D, Michalak
M, Borst J W, Jarmołowski A, Szweykowska-Kulińska Z. (2016). Active 5' splice
sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from
intron-containing genes. Nucleic Acids Res doi: 10.1093/nar/gkw895.
Ponadto opublikowane zostały następujące prace dotyczące biogenezy i działania
miRNA:
1. Stępień A*, Knop K*, Dolata J*, Taube M, Bajczyk M, Barciszewska-Pacak M,
Pacak A, Jarmołowski A, Szweykowska-Kulińska Z. (2016). Post-transcriptional
coordination of splicing and miRNA biogenesis in plants. Wiley Interdiscip Rev
RNA doi: doi: 10.1002/wrna.1403.
2. Barciszewska-Pacak M, Knop K, Jarmołowski A, Szweykowska-Kulińska Z.
(2016). Arabidopsis thaliana microRNA162 level is posttranscriptionally
regulated via splicing and polyadenylation site selection. Acta Biochim Pol epub,
No. 2016_1349, doi: 10.18388/abp.2016_1349.
3. Barciszewska-Pacak M, Milanowska K, Knop K, Bielewicz D, Nuc P, Plewka P,
Pacak AM, Vazquez F, Karłowski W, Jarmołowski A, Szweykowska-Kulińska Z.
(2015). Arabidopsis microRNA expression regulation in a wide range of abiotic
stress responses. Front Plant Sci 6:410.
4. Zieleziński A*, Dolata J*, Alaba S*, Kruszka K*, Pacak A*, Świda-Barteczka A,
Knop K, Stępień A, Bielewicz D, Pietrykowska H, Sierocka I, Sobkowiak L,
Łakomiak A, Jarmołowski A, Szweykowska-Kulińska Z, Karłowski WM. (2015).
mirEX 2.0 - an integrated environment for expression profiling of plant
microRNAs. BMC Plant Biol 15:144.

*równocenne współautorstwo
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REVIEWERS
RECENZENCI

Prof. dr hab. Włodzimierz Krzyżosiak
Department of Molecular Biomedicine
Institute of Bioorganic Chemistry
Polish Academy of Sciences
Poznan, Poland
Zakład Biomedycyny Molekularnej
Instytut Chemii Bioorganicznej
Polska Akademia Nauk
Poznań, Polska

Dr. Andreas Wachter
Center for Plant Molecular Biology
Eberhard Karls Universität Tübingen
Tübingen, Germany
Centrum Biologii Molekularnej Roślin
Uniwersytet Eberharda Karola w Tybindze
Tybinga, Niemcy

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With all of my heart I would like to thank all of the people,
without whom this work would not have been possible.
First of all to Professor Zofia Szweykowska-Kulinska
for giving me the opportunity of scientific development,
help, understanding, support, constructive crticism,
valuable discussions, and fruitful cooperation.
To Professor Artur Jarmolowski
for discussions, help, and scientific support.
To all of my colleagues, co-workers and professors
from the Adam Mickiewicz University in Poznan.
To all of the people from the Department of Gene Expression,
particularly to Agata Stepien, Maria Barciszewska-Pacak and Michal Taube
for their cooperation, invaluable support, inspiration,
and contributions that helped to foster a wonderful work evironment.
To my parents for their support and motivation.
And a special thanks to my husband Michal
for his patience, understanding, and support during my moments of doubt.
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Z całego serca pragnę podziękować Wszystkim,
bez których niniejsza praca nie mogłaby powstać.
Przede wszystkim pani Profesor Zofii Szweykowskiej-Kulińskiej
za możliwość rozwoju naukowego, wszelką pomoc,
zrozumienie, okazane wsparcie, krytyczne uwagi,
cenne merytoryczne dyskusje oraz owocną współpracę.
Panu Profesorowi Arturowi Jarmołowskiemu
za cenne dyskusje, okazaną pomoc oraz wsparcie naukowe.
Wszystkim znajomym, współpracownikom oraz profesorom
z Uniwersytetu im. Adama Mickiewicza w Poznaniu.
Wszystkim doktorom, doktorantom i pracownikom Zakładu Ekspresji Genów,
a w szczególności Agacie Stępień, Marii Barciszewskiej-Pacak oraz Michałowi Taube
za wspaniałą współpracę, nieocenione wsparcie,
inspirację oraz świetną atmosferę pracy.
Moim Rodzicom za wsparcie i motywację.
Szczególne podziękowania składam mojemu Mężowi Michałowi
za cierpliwość, wyrozumiałość oraz wsparcie w chwilach zwątpienia.
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TABLE OF CONTENTS
ABBREVIATIONS ....................................................................................................... 11
SUMMARY ................................................................................................................... 14
STRESZCZENIE .......................................................................................................... 16
INTRODUCTION ........................................................................................................ 18
1 MicroRNAs ......................................................................................................... 18
1.1 MicroRNA biogenesis in plants .............................................................................. 18
1.2 Plant microprocessor complex ................................................................................ 21
1.2.1. DICER LIKE1 (DCL1) ................................................................................... 22
1.2.2. SERRATE (SE) ............................................................................................... 23
1.2.3. HYPONASTIC LEAVES 1 (HYL1) .............................................................. 24
1.2.4. CBC ................................................................................................................. 25
1.2.5. Other components of the plant microprocessor complex ................................ 26
1.3 Distinct characteristics of miRNA pathways in plants and animals ....................... 29
1.4 Plant miRNA gene (MIR) structure ......................................................................... 30
2 Splicing in plants ................................................................................................ 34
2.1 General overview of splicing principles .................................................................. 34
2.2 Alternative splicing ................................................................................................. 38
2.3 Pre-mRNA splicing in plants .................................................................................. 39
3 Polyadenylation mechanism in plants .............................................................. 40
4 Posttranscriptional regulation of miRNA biogenesis ..................................... 43
4.1 Crosstalk between miRNA biogenesis and splicing during maturation of exonic
miRNAs derived from intron-containing genes in plants ....................................... 43
4.2 Biogenesis of miRNAs located within introns of their host genes .......................... 44
4.2.1. Intronic miRNA maturation in mammals ........................................................ 44
4.2.2. Intronic miRNA biogenesis in plants .............................................................. 46
5 Plant miRNAs responsive to biotic and abiotic stress factors ....................... 50
6 Biogenesis of miR319b and miR319b.2 in Arabidopsis thaliana .................... 53
THE AIM OF THE WORK ......................................................................................... 61
MATERIALS AND METHODS ................................................................................. 62
1 Reagents .............................................................................................................. 62
2 Plant material ..................................................................................................... 64
2.1 Plant growth conditions ........................................................................................... 64
2.1.1. Plant culture on MS medium ........................................................................... 64
2.1.2. Plant culture in soil .......................................................................................... 65
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2.2 Plant transformation ................................................................................................ 65
2.2.1. Transformation of Arabidopsis thaliana plants using the floral dip technique 65
2.2.2. Selection of Arabidopsis transgenic lines using BASTA herbicide ................ 66
2.2.3. Transient expression in Nicotiana benthamiana leaves .................................. 66
2.3 Induction of A. thaliana plant response to selected abiotic stresses ....................... 67
2.3.1. Heat stress ....................................................................................................... 67
2.3.2. Salinity stress .................................................................................................. 67
2.3.3. Drought stress .................................................................................................. 67
2.4 Plant treatment with transcription inhibitor – cordycepin (3′-Deoxyadenosine) .... 68
2.4.1. Heat-stressed A. thaliana 17-day-old seedling treatment with cordycepin ..... 68
2.4.2. Agroinfiltrated N. benthamiana leaf disc treatment with cordycepin ............. 68
2.5 Plant treatment with splicing inhibitor – Herboxydiene (Gex-1A) ......................... 69
3 Bacterial cultures ............................................................................................... 70
3.1 Bacteria strains ........................................................................................................ 70
3.2 Bacterial culture media and reagents ...................................................................... 70
3.3 E. coli cell transformation ....................................................................................... 71
3.3.1. Preparation of competent E. coli cells ............................................................. 71
3.3.2. Transformation of E. coli cells using heat-shock method ............................... 72
3.3.3. Transformed E. coli cell growth conditions and selection .............................. 72
3.4 A. tumefaciens cell transformation .......................................................................... 73
3.4.1. Preparation of competent AGL1 cells ............................................................. 73
3.4.2. Transformation of AGL1 cells using electroporation method ........................ 73
3.4.3. Transformed AGL1 cells growth conditions and selection ............................. 73
4 Methods used during work with ribonucleic acid (RNA) .............................. 75
4.1 Northern hybridization ............................................................................................ 75
4.1.1. RNA isolation using TRIzol reagent ............................................................... 75
4.1.2. RNA electrophoresis in agarose gel ................................................................ 76
4.1.3. RNA electrophoresis in polyacrylamide gel ................................................... 77
4.1.4. RNA transfer from gel to membrane ............................................................... 77
4.1.5. Radiolabeling of DNA oligonucleotides ......................................................... 78
4.1.6. Hybridization of radiolabeled oligonucleotides with RNA ............................. 78
4.1.7. Hybridization signal detection ........................................................................ 79
4.2 cDNA preparation ................................................................................................... 80
4.2.1. RNA isolation for cDNA preparation ............................................................. 80
4.2.2. Removing genomic DNA contamination from RNA sample ......................... 80
4.2.3. First-strand cDNA synthesis for RT-PCR/RT-qPCR analysis ........................ 80
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4.2.4. First-strand cDNA synthesis for 5’ RLM-RACE PCR and 3’ RACE PCR
analyses ............................................................................................................. 81
5 Methods used during work with deoxyribonucleic acid (DNA) .................... 82
5.1 Genomic DNA isolation .......................................................................................... 82
5.2 PCR reactions .......................................................................................................... 83
5.2.1. PCR reactions using DreamTaq DNA Polymerase ......................................... 83
5.2.2. PCR reactions using Pfu DNA Polymerase .................................................... 84
5.2.3. Quantitative real-time PCR technique (RT-qPCR) ......................................... 85
5.2.3.1 Gene expression analysis using Power SYBR® Green PCR Master Mix .. 85
5.2.3.2 MiRNA accumulation level analysis using TaqMan® Universal Master
Mix II with UNG ......................................................................................... 86
5.2.4. Site-directed mutagenesis ................................................................................ 87
5.2.5. 5’ RLM-RACE PCR and 3’ RACE PCR analyses ......................................... 88
5.3 DNA electrophoresis in agarose gel ........................................................................ 91
5.4 Genetic construct preparation ................................................................................. 92
5.4.1. DNA purification directly from PCR mixture or extraction from agarose gel 92
5.4.2. Restriction cleavage of DNA .......................................................................... 93
5.4.3. Vector dephosphorylation ............................................................................... 93
5.4.4. DNA ligation ................................................................................................... 93
5.4.5. Plasmid DNA isolation ................................................................................... 94
5.4.6. DNA sequencing ............................................................................................. 94
5.4.7. LR clonase reaction ......................................................................................... 94
5.4.8. Vectors ............................................................................................................ 95
6 Oligonucleotides ................................................................................................. 95
7 Bioinformatic tools and databases .................................................................. 102
7.1 Programs ............................................................................................................... 102
7.2 Databases .............................................................................................................. 103
RESULTS .................................................................................................................... 104
1 Biogenesis of intronic miRNAs in Arabidopsis thaliana ............................... 104
1.1 Characterization of Arabidopsis intronic miRNAs ............................................... 104
1.2 Analysis of miR402 biogenesis in selected abiotic stresses .................................. 108
1.3 Intronic miR402 biogenesis regulation after splicing inhibitor treatment
(Gex-1A) ............................................................................................................... 117
1.4 Active 5’SS controls miR402 biogenesis .............................................................. 122
1.5 The effect of pre-miR402 stem-loop structure localization on miRNA biogenesis
efficiency ............................................................................................................... 129
1.6 The role of SERRATE protein in regulation of intronic miRNA maturation ....... 136
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2 Functional dissection of miR319b and miR319b.2 biogenesis in plants ..... 139
2.1 Preparation of Arabidopsis transgenic lines carrying mutated versions
of MIR319b ........................................................................................................... 139
2.2 Detailed analysis of miR319b and miR319b.2 in selected transgenic lines .......... 143
2.2.1. Analysis of pri-miR319b and mature miRNA levels in MIR319b transgenic
lines ................................................................................................................ 143
2.2.2. Analysis of selected target mRNA levels ...................................................... 145
DISCUSSION .............................................................................................................. 151
1 Biogenesis of intronic miRNAs in Arabidopsis thaliana ............................... 151
1.1 Plant intronic miRNA accumulation depends on developmental stage
or environmental conditions tested ....................................................................... 151
1.2 Plant intronic pri-miRNAs can be transcribed as independent transcription units
from their host pre-mRNAs .................................................................................. 152
1.3 Splicing inhibition stimulates intronic miRNA biogenesis efficiency .................. 154
1.4 Active 5’ splice site controls biogenesis of intronic miR402 ................................ 157
1.5 Selection of polyadenylation site within pri-miRNA transcripts influences plant
intronic miRNA biogenesis efficiency .................................................................. 158
1.6 SERRATE is key player in crosstalk between the plant microprocessor
and spliceosome .................................................................................................... 159
1.7 Co-transcriptional regulation of intronic miRNA biogenesis in plants ................. 164
2 Functional dissection of miR319b and miR319b.2 biogenesis in plants ..... 165
CONCLUSIONS ......................................................................................................... 169
FUTURE PERSPECTIVES ....................................................................................... 170
LITERATURE ............................................................................................................ 175

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